Biosynthesis in vitro of mono- and di-sialosylgangliosides from gangliotetraosylceramide by cultured cell lines and young rat brain. Structure of the products, and activity and specificity of sialosyltransferase

Incubations in vitro of GA1, labeled with 3H in the terminal D-galactopyranosyl group, with nonradioactive CMP-NeuNAc in the presence of homogenates of C21 rat brain glial cells, NIE mouse neuroblastoma cells, 3T3 mouse fibroblasts, SV 40-transformed 3T3 cells, chick embryo fibroblasts, Rous sarcoma virus-transformed chick embryo fibroblasts, and 9-day old rat brain resulted in all cases in the formation in high yield of GM1b, in which the neuraminidase-labile NeuNAc group is linked at O-3 of the terminal D-galactosyl residue, as shown by permethylation studies. No trace of the naturally occurring neuraminidase-stable GM1a was detected in any case. In addition, with NIE cells, and normal and RSV-transformed chick embryo fibroblasts, a disialosylganglioside (GD1) differing from GD1a and GD1b, and bearing only one substituent at O-3 of the terminal D-galactopyranosyl residue was formed. It was also biosynthesized from GM1b and CMP-NeuNAc by NIE and chick embryo cells but not by C21 cells, or rat brain. However, C21 cells and rat brain were capable of synthesizing GD1a from GM1a. Periodate oxidation degraded both NeuNAc groups in GD1 to a 7-carbon fragm:nt, indicating lack of substitution at O-8. GM1b could not be detected as a natural product in rat brain.